Hiroshi Uchida

Stem cell-like properties of the endometrial side population: Implication in endometrial regeneration

Background The human endometrium undergoes cyclical regeneration throughout a womans reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium. Methodology/Principal Findings We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo. Conclusions/Significance These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis.

Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in colorectal cancer

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) is a 7‐transmembrane receptor reportedly expressed in stem cells of the intestinal crypts and hair follicles of mice. Overexpression of LGR5 is observed in some types of cancer; however, there has been no specific assessment in colorectal tumorigenesis. We performed quantitative RT‐PCR for LGR5 expression in 37 representative cancer cell lines, and showed that LGR5 mRNA was frequently overexpressed in colon cancer cell lines. Moreover, LGR5 expression was higher in colon cancer cell lines derived from metastatic tumors compared with those from primary tumors. In clinical specimens, there was significant overexpression of LGR5 in 35 of 50 colorectal cancers (CRCs), and in seven of seven sporadic colonic adenomas, compared with matched normal mucosa. This suggests up‐regulation of LGR5 from the early stage of colorectal tumorigenesis. LGR5 expression showed marked variation among CRC cases and correlated significantly with lymphatic invasion, vascular invasion, tumor depth, lymph node metastasis, and tumor stage (IIIC vs. IIIB). In addition to cancer cells, crypt base columnar cells of the small intestine and colon were shown by in situ hybridization to express LGR5. This is the first report suggesting the involvement of LGR5, not only in early events but also in late events in colorectal tumorigenesis. (Cancer Sci 2010)

Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.

The UDP-Glucose Receptor P2RY14 Triggers Innate Mucosal Immunity in the Female Reproductive Tract by Inducing IL-8

Innate mucosal immune responses, including recognition of pathogen-associated molecular patterns through Toll-like receptors, play an important role in preventing infection in the female reproductive tract (FRT). Damaged cells release nucleotides, including ATP and uridine 5′-diphosphoglucose (UDP-glucose), during inflammation and mechanical stress. We show in this report that P2RY14, a membrane receptor for UDP-glucose, is exclusively expressed in the epithelium, but not the stroma, of the FRT in humans and mice. P2RY14 and several proinflammatory cytokines, such as IL-8, are up-regulated in the endometria of patients with pelvic inflammatory disease. UDP-glucose stimulated IL-8 production via P2RY14 in human endometrial epithelial cells but not stromal cells. Furthermore, UDP-glucose enhanced neutrophil chemotaxis in the presence of a human endometrial epithelial cell line in an IL-8-dependent manner. Administration of UDP-glucose into the mouse uterus induced expression of macrophage inflammatory protein-2 and keratinocyte-derived cytokine, two murine chemokines that are functional homologues of IL-8, and augmented endometrial neutrophil recruitment. Reduced expression of P2RY14 by small interfering RNA gene silencing attenuated LPS- or UDP-glucose-induced leukocytosis in the mouse uterus. These results suggest that UDP-glucose and its receptor P2RY14 are key front line players able to trigger innate mucosal immune responses in the FRT bypassing the recognition of pathogen-associated molecular patterns. Our findings would significantly impact the strategic design of therapies to modulate mucosal immunity by targeting P2RY14.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a womans reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells.

Impairment of Decidualization in SRC-Deficient Mice

Abstract Many signaling events induced by ovarian steroid hormones, cytokines, and growth factors are involved in the process of decidualization of human and rodent endometrium. We have reported previously that tyrosine kinase activation of SRC functionally participates in decidualization of human endometrial stromal cells. To address its essential role in decidualization, we examined, using wild-type and Src knockout mice, whether the process of decidualization was impaired in the absence of SRC. Immunohistochemistry using an antibody specific for the active form of SRC revealed that the active SRC was expressed prominently in the decidualizing stromal cells of the pregnant wild-type mouse. Moreover, the active SRC was upregulated in the uterine horn with artificially stimulated decidual reaction. In comparison with wild-type and Src heterozygous mice, the uterus of Src null mice showed no apparent decidual response following artificial stimulation. Ovarian steroid-induced decidualization in vitro, as determined by morphological changes and expression of decidual/trophoblast prolactin-related protein and prostaglandin-endoperoxide synthase 2 (also known as Cox2), both of which are decidualization markers, did not occur in a timely fashion in endometrial stromal cells isolated from the uteri of SRC-deficient mice compared to those from wild-type and Src heterozygous mice. Our results collectively suggest that SRC is an indispensable signaling component for maximal decidualization in mice.

OCT4 expression in human uterine myometrial stem/progenitor cells

BACKGROUNDnThe transcription factor, octamer-binding transcription factor 4 (OCT4)/POU5F1, is expressed in embryonic stem cells, germ cells and some types of adult stem cells. Human OCT4 encodes two isoforms, OCT4A and OCT4B. While OCT4A plays a crucial role in the maintenance of stem cell properties, including pluripotency, whereas OCT4B does not. We previously reported that human myometrium contains side population cells (myoSP) with a Hoechst 33 342 low-fluorescent profile. These cells exhibit phenotypic and functional characteristics of myometrial stem cells. The objective of this study was to investigate the comparative expression of OCT4 in the stem/progenitor cell population of the human myometrium.nnnMETHODSnHuman myometrial tissue samples were collected from 18 consenting patients who underwent hysterectomy because of benign gynecological diseases. The resultant isolated or cultured myometrial cells and isolated myoSP were subjected to semi-quantitative and real-time RT-PCR analyses, immunoblot analyses and immunohistochemistry.nnnRESULTSnRT-PCR and immunoblot analyses revealed that OCT4 mRNA and OCT4 protein were detectable in some (but not all) myometrial samples. Immunohistochemistry showed that OCT4 protein was confined to the nuclei of relatively few cells in myometrial tissues expressing OCT4 mRNA. OCT4 and OCT4A transcripts, but not those of OCT4B, were more abundant in myoSP than in non-myoSP, as determined by real-time and semi-quantitative RT-PCR analyses.nnnCONCLUSIONSnRelatively few myometrial cells express OCT4 protein. OCT4 mRNA, in particular OCT4A mRNA, is up-regulated in myoSP that have been reported to exhibit stem cell-like properties. Taken together, the present results indicate that the myoSP population is enriched in OCT4 mRNA.

Premature ovarian failure and androgen receptor gene CAG repeat lengths weighted by X chromosome inactivation patterns

The CAG repeat lengths weighted by X-inactivation ratios were significantly shorter in 58 Japanese patients with premature ovarian failure (POF) than in 42 age-matched control females with normal menses. The results suggest that short CAG repeats with a relatively high androgen receptor function may constitute a susceptibility factor for the development of POF.

Review: Human uterine stem/progenitor cells: Implications for uterine physiology and pathology

The human uterus is composed of the endometrial lining and the myometrium. The endometrium, in particular the functionalis layer, regenerates and regresses with each menstrual cycle under hormonal control. A mouse xenograft model has been developed in which the functional changes of the endometrium are reproduced. The myometrium possesses similar plasticity, critical to permit the changes connected with uterine expansion and involution associated with pregnancy. Regeneration and remodeling in the uterus are likely achieved through endometrial and myometrial stem cell systems. Putative stem/progenitor cells in humans and rodents recently have been identified, isolated and characterized. Their roles in endometrial physiology and pathophysiology are presently under study. These stem/progenitor cells ultimately may provide a novel means by which to produce tissues and organs in vitro and in vivo.

Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in gastric cancer

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT‐PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (Pu2009=u20090.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (Pu2009=u20090.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer.